Polo-like kinases (Plks) are a conserved subfamily of Ser/Thr protein kinases that play pivotal roles in cellular proliferation. Since Plk1 overexpression is closely associated with oncogenesis, Plk1 is considered an attractive target for anti-cancer therapy. The polo-box domain (PBD) uniquely found in the C-terminal non-catalytic region of Plks forms a phosphoepitope-binding module for protein-protein interaction. Here, we report the identification of minimal phosphopeptides that specifically interacted with the PBD of Plk1, but not the two closely-related Plk2 and Plk3, with a high affinity. Comparative binding studies and analyses of the crystal structures of the Plk1 PBD in complex with a minimal phosphopeptide (PLHSpT), its derivative PPHSpT, or no peptide, revealed that the C-terminal SpT dipeptide functions as a high affinity anchor, whereas the N-terminal PLH residues are critical for providing both specificity and affinity to the PBD. Glycerol molecule and sulfate anion are functional equivalents to the Ser and the phosphate moiety of the phospho-Thr, respectively. Testing of a minimal phospho-Thr mimetic peptide demonstrated that inhibition of the PBD of Plk1 is sufficient to induce a mitotic arrest and apoptosis. Thus, the mode of PLHSpT binding to the PBD may provide an important template for designing anti-Plk1 therapeutic agents.